Glycosylation is the most common post-translational modification of cell surface and extracellular matrix proteins. The glycoprotein glycans expressed by a given organism comprise complex mixtures reflective of the non-template driven biosynthesis of these posttranslational modifications. The mixtures contain components that, while physiologically relevant, represent a small fraction of the total glycan population. Thus, characterization of glycoproteins has been a challenging task.
Glycoproteins play important roles in many cellular functions, such as cell-adhesion and immune responses. Changes in glycan profiles have been correlated with altered physiological conditions and disease states, such as cancer and rheumatoid arthritis. Therefore, the ability to detect and quantify the glycan components is important.
Glycoproteins may be glycosylated at different glycosylation sites (glycosites). Glycosylation typically occurs at asparagine (N-glycosylation) or serine (O-glycosylation) residues of the glycosites. In addition, at any particular glycosite, different glycans (oligosaccharides) may be attached to that particular glycosite. This structural heterogeneity complicates the study of glycoproteins, particularly the carbohydrates attached to the glycoproteins. Complicating glycan heterogeneity is the fact that the amount of glycoprotein sample available for analysis from biological sources is often limited. For these reasons, mass spectrometry, which is both highly sensitive and able to characterize complex mixtures of compounds, has emerged as the ideal technology for glycan analysis. Unfortunately, glycans have inherently low ionization efficiency.
Because of this, many researchers have attempted to derivatize glycans with chemical compounds that can increase ionization efficiency (see, Hase, S., Precolumn derivatization for chromatographic and electrophoretic analyses of carbohydrates, J. Chromatography A, 1996, 720(1-2): p. 173-182). The most common “tagging” method is reductive amination of the free reducing end (i.e., the open ring aldehyde form) of the glycan with an aromatic compound containing a primary amine. These workflows require addition of reducing agents, the most common of which is sodium cyanoborohydride, in order to stabilize the imine formed during the derivatization reaction. Thus, these strategies involve significant sample handling and purification steps, which increases the likelihood that valuable analyte may be lost.
However, as in reductive amination approaches, the sample preparation and amount of sample handling needed to utilize the R-glycosylamine intermediates for derivatization chemistry are significant, and not compatible with high throughput glycan analysis. Thus, there are currently no experimental protocols that combine the benefits of glycan derivatization (increased ionization efficiency) with rapid sample preparation or minimal sample manipulation.